Figure 4

ALFY is crucial for retinoid-mediated differentiation of AML cells. Different NB4 or HL60 ALFY knock-down cell lines were generated by lentiviral transduction of shRNAs targeting the ALFY mRNA at three different sites (#1-#3) or a mixture of all three shRNAs (M). (a) NB4 shALFY cell lines or control-transduced cells (CTRL) were treated with 1 µM ATRA as indicated for 6 days before the assessment of granulocytic differentiation. Standard deviations are representative of three independent experiments. ALFY knockdown efficiency as well as CSF3R and CEBPE differentiation markers were determined by qPCR. Raw Ct values were normalized to HMBS and to the untreated control cell line using the 2^−∆∆CT method. (b–c) Neutrophil functionality and granulocytic differentiation were determined with the use of a nitroblue tetrazolium (NBT) reduction assay. NB4 control (CTRL) and shALFY cells were stimulated with phorbol myristate acetate (PMA) in the presence of NBT and subjected to the CytoSpin procedure. The percentage of NBT positive cells under ATRA or acitretin treatment was quantified. Differences in colour intensity were not considered. Mann-Whitney-U test: *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001. (d) NB4 control (CTRL) or shALFY transduced cells were treated with 1 µM acitretin for 6 days and qPCR analyses were performed to measure ALFY, CEBPE and GC3SFR mRNAs. Raw Ct values were normalized to HMBS and to the untreated control of day 6 (∆∆Ct). Standard deviations were calculated on three independent experiments. (e-f) NBT assays were performed as described in Fig. 2. The percentage of positive cells was calculated from three independent experiments, comparing control and ALFY depleted cells under acitretin treatment at day 6 (g) HL60 control and shALFY transduced cells were treated with 1 µM ATRA and knockdown efficiency as well as differentiation was assessed by measuring ALFY, CEBPE and CSF3R mRNA expression. Normalization was performed as described in A. Mann-Whitney-U test: *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001.